Haptens, immunogens, antibodies and conjugates to ketamine and its metabolites

ABSTRACT

The invention provides haptens, immunogens comprising such haptens coupled to an antigenicity-conferring carrier material, conjugates comprising such haptens bonded to a labelling agent as well as, antibodies raised against such immunogens and capable of binding with ketamine and its primary metabolite, norketamine.

[0001] The present invention relates to haptens that are useful for thepreparation of immunogens, antibodies and conjugates, for use incompetitive immunoassays for the detection of ketamine[(±)-2-(o-chlorophenyl)-2-(methylamino) cyclohexanone] and its primarymetabolite, norketamine.

[0002] The present invention also relates to a method and kit fordetecting or determining ketamine and norketamine.

[0003] By “detecting” is meant qualitatively analysing for the presenceor absence of a substance.

[0004] By “determining” is meant quantitatively analysing for the amountof a substance.

[0005] Ketamine (see FIG. 1 of the accompanying drawings) is ashort-acting parenteral anaesthetic agent that has been in clinical usefor 30 years. The drug causes profound analgesia at subanaesthetic dosesand lacks the cardiorespiratory depressive effects associated with mostother general anaesthetics. Despite these important clinical advantages,however, disturbing emergence reactions, including delirium andunpleasant dreams, frequently accompany ketamine therapy and limit itsclinical usefulness. Although the origin of these adverse centralnervous system (CNS) side-effects remains obscure, it has beenspeculated that metabolites of ketamine may play an important role sincecirculating concentrations of the parent drug are very low duringemergence from ketamine-induced anaesthesia. Ketamine undergoesextensive metabolism by hepatic enzymes, yielding mainly, byN-demethylation, norketamine (see FIG. 1 of the accompanying drawings).This primary amine is metabolised further by hydroxylation of thecyclohexane ring system at the positions 4, 5 and/or 6 to produce afamily of hydroxylated norketamines, the predominant member of which is6-hydroxynorketamine (see FIG. 1 of the accompanying drawings).

PRIOR ART

[0006] To date, the determination of ketamine and its metabolites inbiological fluids has been based mainly on gas-chromatography-massspectrometry (GC-MS) and HPLC. These chromatographic methods provideexcellent sensitivity and selectivity but require derivatisation ofketamine and its metabolites. These methods are, in addition, too costlyand time-consuming for use as screening tools.

[0007] Specific binding reactions, such as antibody-antigeninteractions, have been used extensively in immunoassays to detect avariety of substances present in biological fluids.

[0008] Thus, for example, radioimmunoassays could be used for thedetermination of ketamine and its metabolites. Radioimmunoassays arevery sensitive, but do require radionuclide tracers, for example ¹²⁵Iand ³H, and in some cases, a preliminary extraction step. There are noknown RIAs for ketamine.

[0009] Enzyme-linked immunosorbent assays (ELISAs) are a nonradioactivealternative that could be used for the qualitative and quantitativedetermination of ketamine and its metabolites. Again, there were noknown ELISAs for ketamine when priority-forming European PatentApplication No. 02075445.3 was filed on Jan. 31, 2002. Since then,Neogen Corporation launched an ELISA kit for ketamine with 100%cross-reactivity to ketamine and 4.6% cross-reactivity to norketamine.It is believed that the Neogen kit uses antibodie and conjugatesdeveloped against a ketamine hapten incorporating at least themethylamino epitope of ketamine.

[0010] EP 0 459 387 A2 (Abbott Laboratories) describes a fluorescencepolarisation assay for phencyclidine and certain phencyclidinederivatives. Phenylcyclohexylamine derivatives are conjugated to carriermaterials to produce immunogens and to fluorescein derivatives toproduce tracers. Antibodies, preferably monoclonal, are generated to theimmunogens. An immunoassay for phencyclidine and phencyclidinederivatives is developed employing the aforementioned antibodies andtracers. This immunoassay exhibits a high degree of specificity tophencyclidine, phencyclidine metabolites and phencyclidine analogs. Itdoes not cross-react significantly with ketamine (see Table 3(c) at page18). Although the phenylcyclohexylamine derivatives exhibit structuralsimilarity to the haptens of the present invention, the absence of achloro group on the phenyl ring and of a carbonyl group on thecyclohexyl ring are both significant, as is borne out by the lack ofcross-reactivity of the Abbott immunoassay with ketamine.

[0011] Owens et al: (‘Antibodies against arylcyclohexylamines and theirsimilarities in binding specificity with the phencyclidine receptor’ (J.Pharmacol. Exp. Ther. (1988), 246(2), 472-478)) describes antibodiesagainst five epitopes of phencyclidine (PCP)-like molecules, todetermine the molecular requirements of arylcyclohexylamine binding tothe PCP receptor. Four haptens are synthesised from derivatives of PCPand the TCP analog, namely PCHP; PCHBA; tPPC and TCHP. The fifth haptenis made from phenylcyclohexylamine: PCHAP. These haptens are conjugatedto BSA to produce immunogens that are used to generate antibodies. Theantibodies produced are highly specific for PCP and some relatedcompounds but exhibit very low cross-reactivity to ketamine (<0.6%)(Table 2, page 475). The hapten PCHAP differs from the haptens of thepresent invention in that there is no chloro group on the phenyl ringand no carbonyl group on the cyclohexyl ring. The absence of both ofthese moieties is, again, significant, as is borne out by the lack ofcross-reactivity to ketamine.

[0012] Owens et al: (‘Molecular requirements for an immunological modelof the phencyclidine receptor’ Sigma and PCP-Like Compounds as MolecularProbes in Biology, 1988) describes the preparation of haptens,immunogens and antibodies to PCP and other arylcyclohexylamines (Table1, page 667). Four haptens are prepared: PCHP; tPPC; PCHBA and PCHAP(see FIG. 1, page 665). The antibodies generated exhibited high levelsof cross-reactivity to PCP and related compounds and low levels ofcross-reactivity to ketamine, as indicated by high IC50 values (Table 1,page 667). Once again, the absence of the chloro and carbonyl groups is,again, significant, as is borne out by the lack of cross-reactivity toketamine.

[0013] Leung et al: (‘Comparative pharmacology in the rat of ketamineand its two principal metabolites, norketamine and(z)-6-hydroxynorketamine’ J. Med. Chem. (1986), 29(11), 2396-2399)describes the synthesis of (z)-6-hydroxynorketamine (a secondarymetabolite of ketamine) from norketamine, the primary metabolite ofketamine. They also perform comparative pharmacology studies in rats onketamine, norketamine and (z)-6-hydroxynorketamine. Leung et al neitherdiscloses nor suggests how to make haptens, immunogens and antibodies toketamine or norketamine.

SUMMARY OF THE INVENTION

[0014] The present invention discloses haptens derivatised with acrosslinker at the N-position of norketamine or at an O-position of ahydroxynorketamine or of a hydroxyketamine.

[0015] The invention also provides immunogens comprising such haptens,coupled to an antigenicity-conferring carrier material, as well as,antibodies raised against such haptens. In addition, the inventionconcerns conjugates comprising such haptens, covalently bonded to adetectable labelling agent.

[0016] The invention also provides methods and kits for detecting ordetermining ketamine and its primary metabolite, norketamine, usingconjugates and antibodies of the present invention.

OBJECT OF THE INVENTION

[0017] It is an object of the invention to overcome some or all of thedisadvantages of the prior art, or to provide an alternative thereto.

DETAILED DESCRIPTION OF THE INVENTION

[0018] In a first aspect, the present invention provides a haptenderivatised with a crosslinker at the N-position of norketamine.

[0019] Preferably, the hapten has the following structural formula:—

[0020] wherein R is a bivalent link and X is a terminal group.

[0021] Most preferably, R comprises a substituted or unsubstituted,straight or branched chain, saturated or unsaturated alkylene moiety;and X comprises a carboxylic acid or an ester thereof; an amine, amaleimide, a halocarboxylic acid or an ester thereof, an aldehyde, adithiopyridyl moiety, a vinylsulphone moiety or a thiocarboxylic acid oran ester thereof.

[0022] Thus, in the formula of the preferred embodiment, the crosslinkercomprises —R—X. The crosslinker, in the broadest aspect of theinvention, may also comprise —R—X.

[0023] More preferably, R is a C₁₋₅, most preferably a C₃, substitutedor unsubstituted, straight chain, saturated alkylene moiety.

[0024] Most preferably, the substituent, if present, is a carbonylgroup.

[0025] Advantageously, X is selected from a carboxylic acid, athiocarboxylic acid, a dithiopyridyl, a maleimide, or an aldehydemoiety. Most advantageously, X is a carboxy (COOH) or thioacetyl(SCOCH₃) moiety.

[0026] Most advantageously, the hapten is selected from the followinghapten derivatives of norketamine: N-(4′-carboxypropyl)norketamine(hapten A); N-(3′-acetylthiopropyl)norketamine (hapten B); andN-(3′-acetylthiopropionamido)norketamine (hapten C)

[0027] These novel haptens are prepared by N-derivatisation, preferablyN-alkylation, of norketamine using the appropriate crosslinkers.

[0028] The resulting haptens can then be further modified at thesefunctionalised positions for conjugation to modified or non-modifiedcarrier materials to provide immunogens for antibody production andconjugates (tracers) that have excellent sensitivity and specificity forthe detection or determination of ketamine and norketamine.

[0029] The present invention discloses the preparation of the firsthapten derivatives of norketamine. These haptens are employed in thepreparation of immunogens by coupling them to modified or non-modifiedantigenicity-conferring carrier materials. The immunogens obtained arethen administered to mammalian hosts to elicit production of specificantibodies, preferably polyclonal antibodies, which are then used todevelop competitive immunoassays for ketamine and norketamine, employinghaptens conjugated to labelling agents as detection reagents.

[0030] The invention also provides an immunogen comprising a hapten ofthe present invention, coupled to an antigenicity-conferring carriermaterial. Preferably, the carrier material is a protein, a proteinfragment, a synthetic polypeptide or a semi-synthetic polypeptide.

[0031] In a still further aspect, the present invention concernsantibodies raised against the immunogen of the present invention, theantibodies being capable of binding with at least one structural epitopeof norketamine or ketamine. Preferably, the structural epitope is thearylcyclohexanone moiety, more preferably the chlorophenylcyclohexanonemoiety, most preferably the o-chlorophenylcyclohexanone moiety, ofketamine and norketamine.

[0032] In a still further aspect, the present invention concernsantibodies having specificity for ketamine characterised by havingcross-reactivity of more than 100%, preferably 100-175%, fornorketamine.

[0033] In a still further aspect, the present invention comprises aconjugate comprising the hapten of the present invention covalentlybonded to a detectable labelling agent. Preferably, the labelling agentis selected from an enzyme, a luminescent substance, a radioactivesubstance, or a mixture thereof More preferably, the labelling agent isan enzyme, preferably a peroxidase, most preferably horseradishperoxidase (HRP). Alternatively, or additionally, the luminescentsubstance may be a bioluminescent, chemiluminescent or fluorescentmaterial.

[0034] The invention further provides a process of preparing theantibodies, the process comprising the steps of immunising an animal,preferably a vertebrate animal, most preferably a mammalian animal, byrepeated administration of an immunogen of the present invention, andcollecting the resulting serum from the immunised animal. Preferably,the process further comprises fixing said serum antibodies to a backingsubstrate, preferably a solid support, most preferably a polystyrenesolid support. Preferably, the antibodies are polyclonal. Alternatively,the antibodies are monoclonal.

[0035] In a still further aspect, the present invention comprises amethod for detecting or determining ketamine and norketamine in asample, the method comprising contacting the sample with the conjugateof the present invention, or a mixture thereof, and with antibodies ofthe present invention, or a mixture thereof; detecting or determiningbound conjugate; and deducing from a calibration curve the presence of,or the amount of, ketamine and norketamine in the sample.

[0036] In a further aspect, the invention includes a kit for detectingor determining ketamine and norketamine, the kit including the conjugateof the present invention, or a mixture thereof; and the antibodies ofthe present invention, or a mixture thereof The kit may optionallyinclude instructions for the use of said conjugates and said antibodiesfor detecting or determining ketamine and norketamine in a sample.

[0037] Preferably, the sample is a solution, such as a biological fluid.More preferably, the sample is serum or urine.

[0038] In the method and kit of the present invention, it is preferredthat the respective crosslinkers (of the immunogen and the conjugate)are different.

[0039] In a further aspect, the present invention involves use of theconjugates of the present invention, or a mixture thereof, with theantibodies of the present invention, or a mixture thereof, to detect ordetermine ketamine and norketamine in samples such as biological fluids.

[0040] Preparation of Haptens

[0041] The haptens N-(4′-carboxypropyl)norketamine A,N-(3′-acetylthiopropyl)norketamine B andN-(3′-acetylthiopropionamido)norketamine C are prepared of FIG. 2 of theaccompanying drawings. The bromination of(o-chlorophenyl)cyclopentylketone 1 with bromine in carbon tetrachloridegives a-bromo-(o-chlorophenyl)cyclopentylketone 2. The bromoketone 2 istreated with liquid ammonia to give1-[(o-chlorophenyl)iminomethyl]cyclopentanol 3. The norketamine 4 isobtained after thermic rearrangement of the alcohol imine 3 at 200° C.The N-alkylation of norketamine 4 by succinic semialdehyde in thepresence of sodium cyanoborohydride gives theN-(4′-carboxypropyl)norketamine (Hapten A). The reaction of norketamine4 with iodopropylthioacetate in ethanolic alkaline solution givesN-(3′-acetylthiopropyl)norketamine (Hapten B). TheN-(3′-acetylthiopropionamido)norketamine (Hapten C) is obtained byreaction of norketamine 4 with 3-acetylthiopropionic acid in DMF in thepresence of EDC.hydrochloride and pyridine.

[0042] The hapten 6-O-(3′-acetylthiopropyl)norketamine D is prepared insix steps according to FIG. 3 of the accompanying drawings. Norketamine4 is converted firstly to the methyl carbamate derivative 5 by reactionwith methyl chloroformate in benzene at reflux in the presence of sodiumcarbonate (Na₂CO₃). The methyl carbamate 5 is treated with lithiumdiisopropylamide (LDA) and trimethylsilyl chloride (TMSC) intetrahydrofuran (THF) at −78° C. to yield the correspondingtrimethylsilyl-enol ether 6. The oxidation of 6 with m-chloroperbenzoicacid (MCPBA) in the presence of Na₂CO₃ gives the N-protected6-hydroxynorketamine 7. The O-alkylation of 6 with allyl bromide inalkaline conditions gives the derivative 8. Removal of themethoxycarbonyl-protecting group of 8 by using trimethylsilyl iodide,followed by reaction with thioacetic acid in chloroform at reflux in thepresence of 2,2′-azobis(2-methylpropionitrile) (AIBN) catalytic yieldsthe hapten 6-O-(3′-acetylthiopropyl)norketamine D in moderate yield.

[0043] Preparation of Immunogens and Conjugates

[0044] Although the haptens of the present invention provide definedstructural epitopes, they are not in themselves immunogenic andtherefore need to be conjugated to carrier materials, which will elicitan immunogenic response when administered to a host animal. Appropriatecarrier materials commonly contain poly(amino acid) segments and includepolypeptides, proteins and glycoproteins. Illustrative examples ofuseful carrier materials are bovine serum albumin (BSA), egg ovalbumin,bovine gamma globulin, thyroxin binding globulin, keyhole limpethaemocyanin (KLH) etc. Alternatively, synthetic poly(amino acids) havinga sufficient number of available amino groups, such as lysine, may beemployed, as may other synthetic or natural polymeric materials bearingreactive functional groups. In particular, carbohydrates, yeasts orpolysaccharides may be conjugated to the hapten to produce an immunogen.

[0045] Each hapten of the present invention can also be coupled to alabelling agent such as an enzyme (for example, horseradish peroxidase),a substance having fluorescent properties or a radioactive label for thepreparation of conjugates (or detection reagents) for use in theimmunoassays. The fluorescent substance may be, for example, amonovalent residue of fluorescein or a derivative thereof.

[0046] In preparing immunogens or conjugates with haptens of the presentinvention where a thiol group is present, such as, for example, haptensB, C and D, maleimide, halo or vinylsulphone groups must first beintroduced to the carrier material or labelling agent (enzyme or label)using heterobifunctional linkers such as:N-(g-maleimidobutyryloxy)succinimide ester (GMBS); succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC);(m-maleimidobenzoyl)-N-hydroxysuccinimide (MBS); succinimidyl4-(p-maleimidophenyl)butyrate (SMPB);N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB); bromoacetylglycineN-hydroxysuccinimide; N-succinimidyl 3-(2-pyridyldithio)propionate(SPDP); or vinylsulphone (Pierce Chemical Company, USA). Thethus-modified carrier material or labelling agent can then be conjugatedvia the thiol groups on the hapten, for example, haptens B, C and D. Forhaptens without a thiol group present, such as hapten A, conjugation isperformed without prior-modification of the carrier material orlabelling agent using standard methods of conjugation such as mixedanhydride, EDC or succinimidyl activation of the hapten.

[0047] In order to confirm that adequate conjugation of hapten tocarrier material has been achieved, prior to immunisation, eachimmunogen is evaluated using matrix-assisted UV laserdesorption/ionisation time-of-flight mass spectroscopy (MALDI-TOF MS).Each of the immunogens of the present invention is suitable forimmunisation, in order to produce antibodies for the detection ofketamine and norketamine.

[0048] General Procedure for MALDI-TOF Analysis of Immunogens.

[0049] MALDI-TOF mass spectrometry was performed using a Voyager STRBiospectrometry Research Station laser-desorption mass spectrometercoupled with delayed extraction. An aliquot of each sample to beanalysed was diluted in 0.1% aqueous trifluoroacetic acid (TFA) tocreate 1 mg/ml sample solutions. Aliquots (1 μl) were analysed using amatrix of Sinapinic acid and bovine serum albumin (Fluka) was used as anexternal calibrant. FIG. 5 of the accompanying drawings shows theanalysis for BSA carrier material. As will be seen, a major signal waspresent which indicates an average protonated mass for this sample ofm/z 66,404. The signal at m/z 33,200 is consistent with the majorcomponent in the doubly-charged form. Further signals were observedincluding m/z 13,605.

[0050] Preparation of Antisera

[0051] In order to generate polyclonal antisera, each immunogen of thepresent invention is mixed with Freund's Adjuvant and the mixture isinjected into a host animal, such as rabbit, sheep, mouse, guinea pig orhorse. Further injections (boosts) are made and serum is sampled forevaluation of the antibody titre. When the optimal titre has beenattained, the host animal is bled to yield a suitable volume of specificantiserum. The degree of antibody purification required depends on theintended application. For many purposes, there is no requirement forpurification, however, in other cases, such as where the antibody is tobe immobilised on a solid support, purification steps can be taken toremove undesired material and eliminate non-specific binding.

[0052] The specific antibodies prepared in this invention are useful asreagents in immunoassays for the detection or determination of ketamineand norketamine in biological fluids. The antibodies of the presentinvention are capable of binding with the arylcyclohexanone moiety,preferably the chlorophenylcyclohexanone moiety, most preferably theo-chlorophenylcyclohexanone moiety, of ketamine and of norketamine.

EXAMPLES Example-1 Preparation ofa-bromo-(o-chlorophenyl)cyclopentylketone 2

[0053] To a solution of (o-chlorophenyl)cyclopentyl ketone 1 (21.0 g,0.1 mol) in 25 ml of anhydrous carbon tetrachloride under inertatmosphere was added, dropwise at 0° C., bromine (16.17 g, 0.1 mol) in100 ml of carbon tetrachloride, over a period of 30 minutes. After allthe bromine had been added, an orange suspension formed. The suspensionwas stirred for 30 minutes at room temperature and then washed using a10% (w/v) solution of sodium bisulfite (2×100 ml). The organic phase wasdried over anhydrous sodium sulfate, filtered and evaporated underreduced pressure to dryness to give thea-bromo-(o-chlorophenyl)cyclopentyl ketone 2 (24.0 g, 83%) as a darkyellow oil, ready for use without further purification in Example 2.

Example-2 Preparation of 1-[(o-chlorophenyl)iminomethyl]cyclopentanol 3

[0054] To a solution of liquid ammonia (100 ml) at −78° C. was addedportionwise over 1 hour period the crude compound 2 (24.0 g, 0.0834mol). The mixture was then stirred for 4 hours and the ammonia allowedto evaporate overnight. The solid residue obtained was suspended intetrahydrofuran and the precipitate was removed by filtration. Thefiltrate was concentrated under reduced pressure and the residueobtained triturated with petroleum ether. The resulting precipitate wasremoved by filtration and dried overnight under P₂O₅ to give the freebase of 1-[(o-chlorophenyl)iminomethyl] cyclopentanol 3 as a white solid(16.8 g, 90%).

[0055] IR (KBr): 3210.9; 1635.9; 763.3 cm⁻¹ NMR¹³C (CDCl₃): 183.4;138.2; 130.3; 130.0; 129.8; 127.2; 84.9; 38.6(2C); 23.6(2C) ppm. M.P.(petroleum ether): 88-90° C.

[0056] The 1-[(o-chlorophenyl)iminomethyl]cyclopentanol hydrochloride 3was prepared by dissolving 20 g (0.089 mol) of the free base of compound3 in 25 ml of isopropanol and to this solution was added 100 ml ofhydrochloric acid (2N) in diethyl ether. The mixture was then stirredfor 10 minutes, and the precipitate obtained was filtered, washed bydiethyl ether and dried to give a white solid of the hydrochloride ofcompound 3 (23.14 g, 100%).

[0057] M.P.: 144-146° C.

Example-3 Preparation of [2-amino-2-(o-chlorophenyl)]cyclohexanone(norketamine) 4

[0058] To 250 ml of Dowtherm-A™ at 200° C. was added in one portion thehydrochloride of compound 3 (20 g, 0.077 mol) and the mixture wasstirred at 200° C. for 15 minutes. The reaction mixture was then cooledat 110° C., and the precipitate formed was removed and discarded byfiltration. The filtrate was diluted in ether and extracted in water(2×200 ml). The combined aqueous phases were washed with ether, madealkaline with 6N sodium hydroxide and extracted in ether (2×150 ml). Thecombined ether layers were washed with water, dried and concentrated todryness. The residue obtained was purified by flash chromatography onsilica gel (70% hexane/30% ethylacetate) to give norketamine free base(13.75 g, 80%) 4 as a colourless oil.

[0059] IR (film): 3381.47; 3310.15; 3064.46; 1939.96; 2864.94; 1712.61and 757.0. NMR¹³C (CDCl₃): 212.85; 140.47; 133.11; 131.08; 129.01;128.36; 127.24; 66.52; 41.35; 39.06; 28.42; 22.23 (FIG. 10).

Example-4 Preparation of N-(4′-carboxypropyl)norketamine-Hapten A

[0060] To a 15% solution of succinic semialdehyde (5 ml, 8.018 mmol,811.7 mg) was added tetrahydrofuran (20 ml), norketamine 4 (1.38 g, 6.17mmol) and sodium cyanoborohydride (388 mg, 6.17 mmol) at 0° C. Themixture was stirred for two hours at room temperature and thin layerchromatography (10% methanol in chloroform) confirmed that the startingmaterial (norketamine 4) was no longer present. To this solution wasadded 20 ml of 1N hydrochloric acid and the reaction mixture was stirredfor an additional 1 hour. Tetrahydrofuran was evaporated under reducedpressure and the remaining aqueous solution was neutralised to pH 7 withsodium hydroxide (1N) and extracted in chloroform (3×50 ml). Thecombined organic layers were washed with 5% sodium bicarbonate solution(50 ml), water (50 ml), dried over anhydrous sodium sulfate, filteredand concentrated to dryness. The residue obtained was purified bychromatography on silica gel (10% methanol in chloroform) to give 0.8 g(38.8%) of N-(4′-carboxypropyl)norketamine (Hapten A) as a whitehygroscopic foam.

[0061] IR (film): 3354.76; 3056.10; 1721.05; 1265.70; 736.68 NMR¹³C(CDCl₃): 208.26; 175.37; 135.33; 134.27; 131.47; 129.85; 129.76; 127.08;70.49; 42.72; 39.02; 38.21; 34.7; 29.01; 24.54; 21.93 (FIG. 11).

Example-5 Preparation of N-(3′-acetylthiopropyl)norketamine-Hapten B

[0062] To a solution of norketamine 4 (2.5 g, 0.011 mol) in 50 ml ofabsolute ethanol was added triethylamine, TEA, (2 ml) andiodopropylthioacetate (2.95 g, 0.0121 mol). The mixture was stirred atreflux for 48 hours (TLC showed that the reaction was complete). Thesolution was evaporated to dryness and the residue obtained was purifiedby chromatography on silica gel (60% hexane/40% ethylacetate) to give1.8 g (48%) of N-(3′-acetylthiopropyl)norketamine (Hapten B) as a clearyellow oil.

[0063] IR (film): 3354.33; 3063.62; 2937.2; 2863.86; 1692.95 (br) and757.97 NMR¹³C (CDCl₃): 209.27; 196.51; 138.84; 134.11; 131.6; 129.52;129.04; 127.06; 70.21; 41.2; 39.83; 39.6; 30.99; 30.92; 28.22; 27.15 and22.2 (FIG. 12).

Example-6 Preparation of N-(3′-acetylthiopropionamido)norketamine-HaptenC.

[0064] To a solution of norketamine 4 (2.5 g, 0.011 mmol) in 10 ml ofanhydrous DMF under nitrogen was added 3-acetylthiopropionic acid (1.79g, 0.012 mol), EDC.hydrochloride (2.53 g, 0.013 mol) and pyridine (1.9g, 0.024 mol). The mixture was then stirred at room temperature for 2hours (TLC indicated no starting material left in the reaction). Themixture was evaporated to dryness, and the crude product obtained waspurified by flash chromatography on silica gel (50% hexane/50%ethylacetate) to give 2.3 g (62%) of theN-(3′-acetylthiopropionamido)norketamine (Hapten C) as a yellow oil.

[0065] IR (film): 3353.79; 3066.59; 2944.2; 2867.78; 1736.06; 1682 (br)and 760.16.

Example-7 Conjugation of Hapten A to BSA—Immunogen A

[0066] 104 mg EDC.hydrochloride was dissolved in water (0.5 ml) andimmediately added to a solution of hapten A (70 mg, 0.22 mmol) in DMF (1ml). After mixing, this solution was added to a solution of BSA (200 mg)in 10 ml of water. Sulfo-NHS (52 mg) was immediately added and thereaction mixture was incubated, with stirring at room temperatureovernight. The mixture was then dialysed against phosphate bufferedsaline (PBS), pH 7.2, (3 changes) for 24 hours, and freeze dried.

[0067] By MALDI-TOF (see FIG. 6 of the accompanying drawings), a signalwas present which indicates an average protonated mass for this sampleof m/z 69,227. These data suggest that an average of 9.7 molecules ofHapten A have been conjugated per molecule of BSA.

Example-8 Preparation of Bromoacetylglycine Modified Bovine SerumAlbumin

[0068] To a solution of BSA (1 g) in 0.1M borate buffer (pH 8.5, 45 ml),cooled to 0° C., was added dropwise N-succinimidyl bromoacetylglycine(0.375 g, 0.13 mmole) in DMF (5 ml). During the addition, the pH wasmaintained at 8.0 and the solution was stirred at 0° C. for one hour.The pH was adjusted to 7 and the solution was dialysed for 24 hours at4° C. against distilled water (3 changes). The solution was then freezedried to give 990 mg of BSA modified by bromoacetylglycine.

[0069] By MALDI-TOF (see FIG. 7 of the accompanying drawings), a majorsignal was present which indicates an average protonated mass for thissample of m/z 73,264. The signal at m/z 36,644 is consistent with themajor component in the doubly-charged form. These data suggest that anaverage of 38.6 lysine groups were modified by the bromoacetylglycine.

Example-9 Conjugation of Hapten B to Bromoacetylglycine ModifiedBSA—Immunogen B

[0070] Hapten B (70 mg) was dissolved in 1 ml of 0.12M potassiumcarbonate in 80% methanol/20% water, the mixture was then set undernitrogen for 10 to 15 minutes (TLC showed no hapten B left and theformation of a new compound with a lower Rf). Phosphate buffer (1 ml-pH7) was added to stop the reaction and the pH was adjusted to 7 by theaddition of a few drops of 0.5M HCl. This solution was added dropwise toa solution of modified BSA (200 mg in 10 ml of water) and the resultingsolution was stirred at 4° C. overnight (protected from light). Thesolution was dialysed against distilled water for 24 hours (3 changes)and freeze-dried.

[0071] By MALDI-TOF (see FIG. 8 of the accompanying drawings), a majorsignal was present which indicates an average protonated mass for thissample of m/z 75,860. The signal at m/z 38,077 is consistent with themajor component in the doubly-charged form. These data suggest that anaverage of 8.8 molecules of Hapten B have been conjugated per moleculeof bromoacetylglycine modified BSA.

Example-10 Conjugation of Hapten C to Bromoacetylglycine ModifiedBSA—Immunogen C

[0072] Hapten C was conjugated to bromoacetylglycine modified BSA usingthe same method used for hapten B (Example-9).

[0073] By MALDI-TOF (see FIG. 9 of the accompanying drawings), a signalwas present which indicates an average protonated mass for this sampleof m/z 76,424. The signal at m/z 38,050 is consistent with the majorcomponent in the doubly-charged form. These data suggest that an averageof 10.2 molecules of Hapten C have been conjugated per molecule ofbromoacetylglycine modified BSA.

Example-11 Conjugation of Hapten A to HRP—Conjugate A

[0074] 10 mg EDC.hydrochloride was dissolved in 800 μl of water andimmediately added to a solution of 2 mg of hapten A in 200 μl of DMF.After mixing, this solution was added to HRP (20 mg) in 1 ml of water.Sulfo-NHS (5 mg) was immediately added and the reaction mixture wasincubated in the dark at room temperature overnight. Excess hapten wasremoved by desalting with 2 PD-10 columns (Pharmacia) in series,pre-equilibrated with PBS at pH 7.2. The hapten-HRP conjugate was thendialysed overnight against 10L of PBS, pH 7.2, at 4° C.

Example-12 Preparation of Bromoacetylglycine Modified HRP

[0075] The preparation of bromoacetylglycine modified HRP was carriedout by the same method outlined in Example 8.

Example-13 Conjugation of Hapten B to Bromoacetylglycine ModifiedHRP—Conjugate B

[0076] 10 mg of Hapten B was dissolved in 0.5 ml of 0.12M potassiumcarbonate solution (80% methanol/20% water). The resulting solution wasset for 10 min at room temperature. 1 ml of 50 mM phosphate buffer, pH7, was added to the solution to stop the reaction and the pH wasadjusted to 7-7.5 using 0.5M HCl. 500 μl of this solution was addeddropwise to a solution of the bromoacetylglycine modified HRP of Example12 (20 mg in 1 ml of water) and the mixture was stirred in the darkovernight at 4° C. The hapten-HRP conjugate was then purified using twoPD-10 columns (Pharmacia Biotechnol), eluted with PBS, pH 7.2, anddialysed overnight against 10L of water.

Example-14 Conjugation of Hapten C to Bromoacetylglycine ModifiedHRP—Conjugate C

[0077] Hapten C was conjugated to bromoacetylglycine modified HRP by thesame method used for hapten B in Example-13.

Example-15 Preparation of Antibodies to Immunogen C, Prepared in Example10

[0078] An aqueous solution of the immunogen prepared in Example 10 wasformulated with Freund's Complete Adjuvant (FCA) to form an emulsionconsisting of 4 mg/ml immunogen in 50% (v/v) FCA. Three sheep wereimmunised with this emulsion (10 immunisation), 0.25 ml beingsubcutaneously injected at each of four sites in the flank of eachanimal. The next immunisation (boost 1) contained 2 mg/ml immunogen andsubsequent immunizations (boosts 2 to 11) contained 1 mg/ml immunogen.All boosts were emulsified in 50% (v/v) Freund's Incomplete Adjuvant(FIA) and were administered in the same manner as the 10 immunisation,at monthly intervals for 1 year. Blood sampling took place 7 to 14 daysafter each boost. Each sample was processed to produce antiserum, whichwas further purified by caprylic acid and ammonium sulfate precipitationto yield an immunoglobulin G (IgG) fraction. The IgG fraction wasevaluated by competitive ELISA microtiter plate assay, as described inExample 16 below.

Example-16 Development of a Competitive ELISA for Ketamine andNorketamine

[0079] The wells of an enhanced binding 96 well polystyrene microtiterplate were coated with the IgG fraction of the antiserum raised toimmunogen C (hapten C-BSA) (Example 10), diluted in 10 mM Tris, pH 8.5(125 μl/well). The appropriate antibody coating dilution was determinedusing standard ELISA checkerboard techniques. The plate was incubatedfor 2 hours at 37° C., washed 4 times with Tris buffered salinecontaining Tween 20 (TBST) and tapped dry. Standard solutions ofketamine and norketamine were prepared in TBST at 0, 0.1, 0.5, 1, 5, 25,100 and 500 ng/ml and 50 μl of each was added to the appropriate wells(FIG. 4). 75 μl of conjugate A (hapten A-HRP) (Example 11), diluted inTris buffer (pH 7.2) containing EDTA, D-mannitol, sucrose, thimerosaland BSA, was added to each of the wells, as shown in FIG. 4. Theappropriate dilution of conjugate was also determined using standardELISA checkerboard techniques. The plate was incubated at 37° C. for 2hours. Excess unbound conjugate was removed by washing 6 times over a 10minute period with TBST.

[0080] 125 μl of tetramethylbenzidine (TMB) substrate solution was addedto each well of the plate that was then incubated for 15 to 20 minutesin the dark at room temperature. The reaction was terminated by additionof 125 μl 0.2M H₂SO₄ to each well. The absorbance was then measured at450 nm using a microtiter plate reader. The data generated in the assayis presented in Table 1 below. TABLE 1 Data generated from a competitivemicrotiter plate assay for ketamine and norketamine, employing antiserumraised to immunogen C (hapten C-BSA) (Example 10) and conjugate A(hapten A-HRP) as detection reagent (Example 11). Standard ConcentrationKetamine Norketamine ng/ml A₄₅₀ % B/B₀ A₄₅₀ % B/B₀ 0 2.194 100.0 2.121100.0 0.1 2.02 92.1 1.843 86.9 0.5 1.817 82.8 1.416 66.8 1 1.489 67.91.368 64.5 5 0.935 42.6 0.961 45.3 25 0.548 25.0 0.482 22.7 100 0.40418.4 0.320 15.1 500 0.254 11.6 0.222 10.5 IC₅₀ 2.9 ng/ml 2.1 ng/ml % CR100.0 138.1

Example 17 Cross Reactivity of the Competitive ELISA for Ketamine andNorketamine

[0081] In order to determine the specificity of the competitive ELISAfor ketamine and norketamine, standard solutions were prepared in TBSTat 0, 0.1, 0.5, 1, 5, 25, 100 and 500 ng/ml. Employing each series ofstandards in the ketamine competitive ELISA, calibration curves weregenerated and these were used to determine the cross-reactivity of theimmunoassay with these substances. The results of this study arepresented in Table 2, cross-reactivity of potential cross-reactantsphencyclidine and dextromethorphan being calculated according to thefollowing formula:

%CR=IC _(50, Ketamine) /IC _(50, CR)×100

[0082] Where %CR is the percentage cross-reactivity, IC_(50, Ketamine)is the concentration of ketamine that causes 50% displacement of signaland IC_(50, CR) is the concentration of potential cross-reactant thatcauses 50% displacement of signal. TABLE 2 Cross reactivity of thecompetitive ELISA for ketamine and norketamine Cross-ReactantIC_(50, CR) (ng/ml) % CR Phencyclidine >500.0 <0.58Dextromethorphan >500.0 <0.58

1. A hapten derivatised with a crosslinker at the N-position ofnorketamine.
 2. The hapten of claim 1, wherein the hapten has thefollowing structural formula:—

wherein R is a bivalent link and X is a terminal group.
 3. The hapten ofclaim 2, wherein R is selected from the group comprising a substitutedor unsubstituted, straight or branched chain, saturated or unsaturatedalkylene moiety; and X is selected from the group comprising acarboxylic acid or an ester thereof; an amine, a maleimide, ahalocarboxylic acid or an ester thereof, an aldehyde, a dithiopyridylmoiety, a vinylsulphone moiety or a thiocarboxylic acid or an esterthereof.
 4. The hapten of claim 3, wherein R is a C₁₋₅ substituted orunsubstituted, straight chain, saturated alkylene moiety.
 5. The haptenof claim 4, wherein R is a C₃ substituted or unsubstituted, straightchain, saturated alkylene moiety.
 6. The hapten of claim 3, wherein X isselected from a carboxylic acid, a thiocarboxylic acid, a dithiopyridyl,a maleimide, or an aldehyde moiety; advantageously, X is a carboxy(COOH) or thioacetyl (SCOCH₃) moiety.
 7. The hapten of claim 6, whereinX is selected from a carboxy (COOH) or thioacetyl (SCOCH₃) moiety. 8.The hapten of claim 1 wherein the hapten is selected from the followinghapten derivatives of norketamine: N-(4′-carboxypropyl)norketamine;N-(3′-acetylthiopropyl)norketamine; andN-(3′-acetylthiopropionamido)norketamine.
 9. The hapten of claim 2,wherein the hapten is selected from the following hapten derivatives ofnorketamine: N-(4′-carboxypropyl)norketamine;N-(3′-acetylthiopropyl)norketamine; andN-(3′-acetylthiopropionamido)norketamine.
 10. An immunogen comprisingthe hapten of claim 1, coupled to an antigenicity-conferring carriermaterial.
 11. An immunogen comprising the hapten of claim 2, coupled toan antigenicity-conferring carrier material.
 12. The immunogen of claim10 wherein the carrier material is selected from a protein, a proteinfragment, a synthetic polypeptide or a semi-synthetic polypeptide. 13.Antibodies raised against the immunogen of claim 10, the antibodiesbeing capable of binding with at least one structural epitope ofnorketamine or ketamine.
 14. Antibodies raised against the immunogen ofclaim 11, the antibodies being capable of binding with at least onestructural epitope of norketamine or ketamine.
 15. The antibodies ofclaim 13, wherein the structural epitope is an arylcyclohexanone moiety.16. The antibodies of claim 15, wherein the structural epitope is achlorophenylcyclohexanone moiety.
 17. A conjugate comprising the haptenof claim 1 covalently bonded to a detectable labelling agent.
 18. Aconjugate comprising the hapten of claim 2 covalently bonded to adetectable labelling agent.
 19. A conjugate comprising the hapten ofclaim 1 covalently bonded to a detectable labelling agent, the labellingagent being selected from an enzyme, a luminescent substance, aradioactive substance, or a mixture thereof.
 20. A conjugate comprisingthe hapten of claim 2 covalently bonded to a detectable labelling agent,the labelling agent being selected from an enzyme, a luminescentsubstance, a radioactive substance, or a mixture thereof.
 21. A processof preparing the antibodies of claim 13, the process comprising thesteps of immunising an animal by repeated administration of theimmunogen of claim 10, and collecting the resulting serum from theimmunised animal.
 22. A process of preparing the antibodies of claim 13,the process comprising the steps of immunising a vertebrate animal byrepeated administration of the immunogen of claim 10, and collecting theresulting serum from the immunised animal.
 23. A process of preparingthe antibodies of claim 13, the process comprising the steps ofimmunising a mammalian animal by repeated administration of theimmunogen of claim 10, and collecting the resulting serum from theimmunised animal.
 24. A process of preparing the antibodies of claim 14,the process comprising the steps of immunising an animal by repeatedadministration of the immunogen of claim 11, and collecting theresulting serum from the immunised animal.
 25. A process of preparingthe antibodies of claim 14, the process comprising the steps ofimmunising a vertebrate animal by repeated administration of theimmunogen of claim 11, and collecting the resulting serum from theimmunised animal.
 26. A method for detecting or determining ketamine andnorketamine in a sample, the method comprising contacting the samplewith the conjugate of claim 17, or a mixture thereof, and with theantibodies of claim 13, or a mixture thereof; detecting or determiningbound conjugate; and deducing from a calibration curve the presence of,or the amount of, ketamine and norketamine in the sample.
 27. A methodfor detecting or determining ketamine and norketamine in a sample, themethod comprising contacting the sample with the conjugate of claim 18,or a mixture thereof, and with the antibodies of claim 14, or a mixturethereof; detecting or determining bound conjugate; and deducing from acalibration curve the presence of, or the amount of, ketamine andnorketamine in the sample.
 28. A method for detecting or determiningketamine and norketamine in a sample, the method comprising contactingthe sample with the conjugate of claim 19, or a mixture thereof, andwith the antibodies of claim 15, or a mixture thereof; detecting ordetermining bound conjugate; and deducing from a calibration curve thepresence of, or the amount of, ketamine and norketamine in the sample.29. A method for detecting or determining ketamine and norketamine in asample, the method comprising contacting the sample with the conjugateof claim 20, or a mixture thereof, and with the antibodies of claim 16,or a mixture thereof; detecting or determining bound conjugate; anddeducing from a calibration curve the presence of, or the amount of,ketamine and norketamine in the sample.
 30. A kit for detecting ordetermining ketamine and norketamine, the kit including the conjugate ofclaim 17, or a mixture thereof; and the antibodies of claim 13, or amixture thereof.
 31. A kit for detecting or determining ketamine andnorketamine, the kit including the conjugate of claim 18, or a mixturethereof; and the antibodies of claim 14, or a mixture thereof.
 32. A kitfor detecting or determining ketamine and norketamine, the kit includingthe conjugate of claim 19, or a mixture thereof; and the antibodies ofclaim 15, or a mixture thereof.
 33. A kit for detecting or determiningketamine and norketamine, the kit including the conjugate of claim 20,or a mixture thereof; and the antibodies of claim 16, or a mixturethereof.
 34. Use of the conjugates of claim 17, or a mixture thereof,with the antibodies of claim 13, or a mixture thereof, to detect ordetermine ketamine and norketamine in samples such as biological fluids.35. Use of the conjugates of claim 18, or a mixture thereof, with theantibodies of claim 14, or a mixture thereof, to detect or determineketamine and norketamine in samples such as biological fluids.
 36. Useof the conjugates of claim 19, or a mixture thereof, with the antibodiesof claim 15, or a mixture thereof, to detect or determine ketamine andnorketamine in samples such as biological fluids.
 37. Use of theconjugates of claim 20, or a mixture thereof, with the antibodies ofclaim 16, or a mixture thereof, to detect or determine ketamine andnorketamine in samples such as biological fluids.